1. Field of the Invention
The present invention relates generally to cellular biochemistry and molecular biology of cationic amino acid transporter proteins. More particularly, the present invention relates to uses for a cationic amino acid transporter protein.
2. Description of the Related Art
Cationic amino acids such as arginine are important in mammalian cellular and tissue function. Biochemically distinct systems mediate their transport into and out of cells; two families of cloned mammalian cationic amino acid transporters have been reported.
Apart from protein synthesis, the cationic amino acid arginine has other crucial roles in cellular processes. Arginine and its transport are essential for the regulated production of nitric oxide (NO). Nitric oxide synthesis requires transport of external arginine into such cells as macrophages, cardiac myocytes, vascular smooth muscle cells and astrocytes. Arginine is the sole precursor for the synthesis of nitric oxide as the amino group donor for nitric oxide, a reaction catalyzed by a family of nitric oxide synthases (NOS). The enzymes nNOS (neural NOS) and eNOS (endothelial NOS) are constitutively expressed in brain and endothelial cells. They transiently produce small amounts of nitric oxide regulated by Ca2+ flux. Inducible NOS (iNOS) is expressed in response to specific cellular signals and produces large amounts of NOS over a sustained period of several days. iNOS activity is rate limited by L-arginine transport. In contrast, nNOS and eNOS activity is independent of L-arginine transport.
On a physiological level, nitric oxide is the most potent vasodilator known and is required for a variety of cellular functions. For example, the cytotoxic activity of macrophages is dependent on nitric oxide. The production of nitric oxide in the vascular endothelium regulates blood pressure, and nitric oxide is a neurotransmitter. Nitric oxide has beneficial biological functions that serve a variety of physiological processes, however, nitric oxide also has less salutary effects. Nitric oxide is unstable and it inhibits enzymes. Intracellular nitric oxide is a highly reactive free radical that reacts with other free radicals, molecular oxygen and heavy metals. Persistent high concentrations of nitric oxide can cause DNA damage.
The role of nitric oxide in pathophysiology is thus suggested, but its precise dimensions are not clear. Although nitric oxide might in some way modulate tumor development, it has been unclear whether it inhibits or stimulates tumor growth, angiogenesis or metastasis.
With respect to the role of nitric oxide in cancer, in particular breast cancer, it has been observed that breast cancer cell lines, human breast cancer cells and mouse mammary tumor cell lines produce nitric oxide in amounts that correlate with tumor grade. Breast cancer tissue samples have been shown to express iNOS in the infiltrating macrophages of the tumor. It has been shown recently that S-nitroso-N-acetyl-DL-penicillamine [a nitric oxide-releasing compound] changes the conformation of recombinant wild-type murine p53 protein and the behavior of p53 protein in CF7 human breast cancer cells. Recent work has shown that p53 expression down-regulates iNOS expression. Additionally, nitric oxide directly affects the regulation of p53 gene expression as well as the conformation and activity of the p53 protein. It is possible that nitric oxide induces mutations in p53 that abrogate iNOS regulation, and nitric oxide induced mutations in p53 could contribute to cell transformation. Data suggests that excess nitric oxide produced in inflamed tissues might play a role in carcinogenesis by impairing the tumor suppressor function of p53.
Further, the role of nitric oxide in angiogenesis has great relevance to breast cancer and vascular density measurement is now widely recognized as a prognostic indicator in breast cancer. In the early 90s, reports began to associate the production of nitric oxide with angiogenesis. Since then, there have been a large series of conflicting reports indicating that nitric oxide stimulates or inhibits angiogenesis. Recently, it has been established that hypoxia, such as that found in early tumors, induces vascular endothelial growth factor (VEGF), angiogenesis and iNOS. Further, very recent work has clearly established that nitric oxide is required for vascular endothelial growth factor to mediate angiogenesis in vitro.
To further elucidate the biochemical underpinnings of nitric oxide synthesis, the identification of cationic amino acid transporter cDNAs was an important first step. The role of regulated transport in the functioning of cell types such as macrophages and others can thus be better understood and manipulated. The majority of arginine transport in most cells and tissues is mediated by a transport system apparently encoded by three genes: Cat1, Cat2 and Cat3. Cat1 and Cat2 genes encode similar proteins, CAT1 and CAT2 (e.g. MacLeod, C. L., Biochem. Soc. Trans., 24:846-852 (1996)), comprising a functionally defined, transport system (y+), which facilitates the transport of the cationic amino acids lysine, arginine and ornithine in a sodium-independent manner. The Cat2 gene encodes two protein isoforms, CAT2 and CAT2a, results of mutually exclusive alternate splicing. The CAT2a protein exhibits a significantly lower (10-fold) apparent affinity for its substrate than either CAT1 or CAT2.
The various CAT transcripts are expressed in distinct patterns. CAT1 transcripts are constitutively and nearly ubiquitously expressed in normal tissues and cell lines. The adult liver does not express CAT1, but exclusively expresses the CAT2a isoform. The expression of CAT2 and CAT2a is much more limited than that of CAT1. Transcripts are abundant only in liver, skeletal muscle, and stomach as well as activated macrophages. Both Cat1 and Cat2 genes are inducible in a variety of circumstances. Expression of the newly reported Cat3 gene is limited to the brain.
With respect to the role of CATs in immune function, resting splenocytes (largely comprised of quiescent T- and B-cells) express predominantly CAT1 mRNA, yet exhibit extremely limited L-arginine and L-lysine transport. Following mitogen or antigen mediated T-cell activation, however, extracellular L-arginine is needed and both CAT1 and CAT2 transcripts rapidly accumulate.
Similarly, quiescent macrophages express only CAT1 mRNA, which steadily decreases for 24 hours following activation. In contrast, CAT2 expression is undetectable until activation. Following activation with lipopolysaccharide (LPS) and IFN-xcex3, macrophages increase system y+ transport following activation to provide these cells with adequate L-arginine for nitric oxide synthesis. At the same time, iNOS is induced. Such observations reflect macrophage requirements for extracellular L-arginine transport for nitric oxide synthesis via the inducible form of nitric oxide synthase (iNOS). It would thus appear that in activated macrophages, CAT2 mediated arginine transport regulates the arginine:nitric oxide pathway.
Although the functions of the CAT genes may be seen as overlapping, they are responsive to different cellular signals. Cat2 gene expression is inducible and highly tissue-specific, while Cat1 is widely expressed and believed to be a housekeeping gene. CAT1 knockout mice are not viable, are runted, anemic and die within hours of birth. CAT2, but not CAT1, mRNA is induced in response to specific cellular activators in several cultured cell systems and in vivo. For example, in macrophages and in mammary cell lines an induction of CAT2 mRNA is seen in response to LPS and IFN-xcex3 coordinately with iNOS. CAT1 mRNA levels decrease or remain low and unaltered. The co-induction of CAT2 and iNOS has been observed in such cell types as macrophages, vascular smooth muscle cells, astrocytes and other glial cells, liver and numerous others.
The prior art lacks effective and specific mechanisms to precisely inhibit cationic amino acid transport in specific cell or tissue types and thereby curtail nitric oxide production in these cells or tissues. The present invention fulfills this long-standing need and desire in the art.
The identification and manipulation of the precise L-arginine transporter involved in nitric oxide production provides new opportunities for therapeutic intervention. The instant invention delineates CAT2 involvement in L-arginine transport and nitric oxide synthesis in various cell types and pathological conditions. It further provides novel and powerfully precise means to regulate this important substance. The instant invention strongly demonstrates a stimulatory role for nitric oxide in tumor growth. Data from studies using transgenic mice with an iNosxe2x88x92/xe2x88x92 genotype demonstrate that lack of iNOS inhibits tumor formation.
One object of the present invention is to provide an antisense methodology for inhibiting cationic amino acid transport comprising the step of administering to a human or a non-human mammal an effective dose of the antisense oligonucleotides of the present invention. Thus, the present invention provides an antisense oligonucleotide directed against CAT2 mRNA. A representative antisense oligonucleotide has the nucleotide sequence: GTAGGCTGAAACCCTGTCCTTGC (SEQ ID No. 2). Further, the present invention provides a pharmaceutical composition comprising the antisense oligonucleotide directed against CAT2 mRNA and a physiologically acceptable carrier.
In another embodiment of the present invention, there is provided a method of treating breast cancer in an individual in need of such treatment, comprising the step of administering to said individual an effective dose of the antisense oligonucleotide directed against CAT2 mRNA.
In another embodiment of the present invention, there is provided a method of treating breast cancer in an individual in need of such treatment, comprising the step of administering to said individual an effective dose of anti-CAT2 antibody.
In one embodiment of the present invention, there is provided a method of treating a pathophysiological state in a human or a non-human mammal, wherein said state is characterized by production of an undesirable level of nitric oxide, comprising the step of administering an effective dose of the antisense oligonucleotides or antibody of the present invention.
In another embodiment of the present invention, there is provided a method of treating a pathophysiological state in a mammal, wherein said state is characterized by production of an undesirable level of nitric oxide, comprising the step of administering an effective dose of a substance designed to specifically block the capacity of CAT2 cell surface protein to transport arginine such as an anti-CAT2 antibody to block cationic amino acid transport and concomitant nitric oxide synthesis. This can be accomplished by using cell lines from a CAT2 deficient and wild type mice to screen for substances that block transport.
In yet another embodiment of the present invention, there is provided a transgenic animal lacking exon 2 of the CAT2 gene and lacking any function of the CAT2 gene.
In another embodiment of the present invention, there is provided a cell line derived from the transgenic animal disclosed herein.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.